It is well established that cells are frequently exposed to changing levels of mechanical tension, and that they respond to tension through signaling pathways that affect their cytoskeletal organization, their cell shape and often their gene expression. Tension is usually transmitted through membrane-spanning cell adhesion molecules that either mediates attachment to the extracellular matrix or to other cells. At their cytoplasmic face these adhesion molecules connect with the cytoskeleton. Tension on adhesion molecules can derive from external sources or be generated by a cell's own actomyosin contractile system. Many of the signaling pathways initiated by tension on adhesion molecules converge to regulate Rho family GTPases, which in turn, influence many aspects of cell behavior. The goal of this grant is to understand how mechanical tension on different adhesion molecules signal to Rho GTPases, focusing on integrins, cadherins and the tight junction protein JAM-A. Applying tension to these adhesion molecules via magnetic beads, we will identify how tension activates pathways that regulate the activities of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). Within a tissue as tension increases on one cell there is often a parallel decrease in tension on another, and cells need to be able to respond to both increasing and decreasing levels of mechanical tension. Consequently, we will also investigate how release of tension affects the signaling pathways that regulate Rho GTPase activity. With adherens junctions and tight junctions, tension can either enhance barrier function and junction assembly, or it can increase permeability and promote disassembly. We will test the hypothesis that these divergent effects of tension on junctions derive from the influence of additional signaling pathways that modulate the specific response to tension. Mechanical tension is also relayed from the cell surface to the nucleus, which is linked to the cytoskeleton via multiple connections. We have shown recently that isolated nuclei stiffen in response to tension and that blocking this response affects cytoskeletal organization as well as cell migration. Building on these findings, as well as on the work of others showing similar consequences from disrupting the links between the nucleus and the cytoskeleton, we will explore how the nucleus contributes to the overall tension within the actin cytoskeleton, thereby influencing the activities of Rho GTPases.